We study the mechanism and regulation of protein synthesis in eukaryotic cells. Of special interest are the regulation of protein synthesis by GTP-binding (G) proteins and protein phosphorylation. In addition, we are studying unusual post-translational modifications of the factors that assist the ribosome in synthesizing proteins. The first step of protein synthesis is binding the initiator Met-tRNA to the small ribosomal subunit by the factor eIF2. The eIF2 is composed of three subunits including the G protein eIF2gamma. During translation initiation, the GTP bound to eIF2gamma is hydrolyzed to GDP, and the factor eIF2B recycles eIF2-GDP to eIF2-GTP. Phosphorylation of eIF2alpha on serine 51 coverts eIF2 into an inhibitor of eIF2B. Among the family of eIF2alpha kinases are GCN2 (activated by amino acid starvation), PKR (activated by double-stranded RNA in virally infected cells), PERK (activated by ER stress), and HRI (activated under conditions of low heme). Our structure-function studies on eIF2 have provided insights into human disease. Protein synthesis plays a critical role in learning and memory in model systems, and our studies have linked a human X-linked intellectual disability (XLID) syndrome to altered function of eIF2. In previous studies with collaborators in Israel and Germany, we characterized a human XLID syndrome characterized by intellectual disability and microcephaly. The patients carry a mutation in the EIF2S3 gene encoding eIF2gamma, and genetic and biochemical studies revealed that the mutation disrupts eIF2 complex integrity and translation start codon selection. Over the past year, working with collaborators in Germany, Slovakia, and at Walter Reed National Military Medical Center, we characterized two additional mutations in eIF2gamma found in patients exhibiting intellectual disability, epilepsy, hypogonadism and hypogenitalism, microcephaly, and obesity. Based on this constellation of phenotypes, the disease has been termed MEHMO syndrome, and we now conclude that MEHMO syndrome is caused by mutations in EIF2S3. Our studies of a yeast model of the MEHMO syndrome mutations in eIF2gamma reveal impaired eIF2 function, altered translational control of specific mRNAs, and reduced stringency of translation start site selection. Consistent with these properties, the Integrated Stress Response, a translational regulatory response typically associated with eIF2alpha phosphorylation, is induced in patient cells. Based on our studies we propose that more severe EIF2S3 mutations cause the full MEHMO phenotype, while less deleterious mutations cause a milder form of the syndrome with only a subset of symptoms. When expressed in yeast, human PKR phosphorylates eIF2alpha causing inhibition of protein synthesis and yeast cell growth. To subvert the anti-viral defense mediated by PKR, viruses produce inhibitors of the kinase. We are characterizing mutations in PKR that confer resistance to the poxviral E3L inhibitor. In a related project, we characterized the insect baculovirus PK2 protein, an eIF2alpha kinase inhibitor that structurally mimics the C-terminal lobe of a protein kinase domain. Together with collaborators in Canada and Japan, we revealed that PK2 targets an insect HRI-like kinase through an unusual lobe-swapping mechanism to generate a nonfunctional pseudokinase complex. In our final two projects, we are studying the translation factors eEF2 and eIF5A. These proteins both function in translation elongation and interestingly carry novel post-translational modifications. In collaboration with researchers at the MRC in Cambridge, we are studying the translation elongation factor eEF2. The eEF2, like its bacterial ortholog EF-G, promotes translocation of tRNAs and mRNA on the ribosome following peptide bond formation. A conserved histidine residue in eEF2 is post-translationally modified to diphthamide through the action of 7 non-essential proteins. The function of diphthamide and rationale for its evolutionary conservation are not well understood, and the only known function of diphthamide is to serve as a substrate for inactivation by diphtheria toxin. Using a yeast in vitro reconstituted assay system, we found that the absence of diphthamide had no impact on peptide synthesis in assays employing the canonical initiation pathway. However, loss of diphthamide significantly impaired peptide synthesis in assays directed by the cricket paralysis virus (CrPV) internal ribosome entry site (IRES). As the CrPV IRES relies on two pseudotranslocation events prior to the first peptide bond formation, we propose that the precise phasing of pseudotranslocation is dependent on the diphthamide modification on eEF2. Consistent with this interpretation, structural studies by our collaborators revealed that diphthamide is positioned to break decoding interactions between conserved rRNA bases and the tRNA-mimicking pseudoknot I of the CrPV IRES. Thus, our studies provide the first evidence that diphthamide plays a role in protein synthesis, and we propose that it functions to disrupt the decoding interactions of rRNA in the A-site and to maintain codon-anticodon interactions as the A-site tRNA is translocated to the P site. Finally, the translation factor eIF5A is the sole protein containing the unusual amino acid hypusine. Using molecular genetic and biochemical studies, we showed that eIF5A promotes translation elongation, and that this activity is dependent on the hypusine modification. We also showed that eIF5A from yeast, like its bacterial ortholog EF-P, stimulates the synthesis of proteins containing runs of consecutive proline residues. Consistent with these in vivo findings, we showed that eIF5A was critical for the synthesis of polyproline peptides in reconstituted yeast in vitro translation assays, and using directed hydroxyl radical probing experiments we mapped eIF5A binding near the E site of the ribosome. Working with x-ray crystallographers in France, we obtained the crystal structure of eIF5A bound to the yeast 80S ribosome. The structure revealed interactions between eIF5A and conserved ribosomal proteins and rRNA bases. Moreover, eIF5A occupies the E site with the hypusine residue projecting toward the acceptor stem of the P-site tRNA. In related studies, we reported the structure of a diproline-tRNA analog bound to the ribosome, revealing that proline affects nascent peptide positioning in the ribosome exit tunnel. Over the last year, we have continued our studies on eIF5A. In collaboration with researchers at Johns Hopkins University, we reported that eIF5A functions globally to promote both translation elongation and termination. Moreover, exploiting our in vitro reconstituted assay system from yeast, we used misacylated tRNAs to show that the imino acid proline and not tRNA(Pro) imposes the requirement for eIF5A. In addition, we found that the more flexible proline analog azetidine-2-carboxylic acid relaxes the eIF5A requirement for peptide synthesis. We also found that eIF5A could functionally substitute for polyamines to stimulate general protein synthesis. Taken together, our studies support a model in which eIF5A and its hypusine residue function to reposition the acceptor arm of polyprolyl-tRNA in the P site to alleviate stalling and that the body of eIF5A functions like polyamines to enhance general protein synthesis. Finally, in ongoing studies, we have linked eIF5A to the regulation of polyamine metabolism in mammalian cells. Synthesis of antizyme inhibitor (AZIN1), a positive regulator of polyamine synthesis, is inhibited by polyamines. We have found that translational control of AZIN1 synthesis relies on polyamine inhibition of eIF5A function. Thus, eIF5A functions generally in protein synthesis and modulation of eIF5A function by polyamines can be exploited to regulate specific mRNA translation.